Summary of Work: We are examining the relationship between the X ray crystal structures of E. coli DNA polymerase I Klenow fragment and mammalian DNA polymerase beta and their kinetic properties, DNA binding affinity, processivity and fidelity. This year, we completed studies of the fidelity of 26 mutant derivatives of Klenow fragment with amino acid substitutions in the polymerase domain. The results suggest that fidelity at the polymerase active site depends on highly specific enzyme?substrate interactions and is not easily perturbed. Four novel base-substitution mutators were identified: R668A, R682A, E710A and N845A. Each substitutes a conserved amino acid near the polymerase active site and each has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710 and Asn-845 contribute to fidelity in different ways. The latter three cause decreased fidelity at the nucleotide insertion step, while R668A results in lower fidelity in nucleotide insertion and mismatch extension. To better understand the fidelity of base excision repair of DNA lesions, this year we also focused on studies of wild-type human pol beta. This enzyme exhibits low fidelity when filling short gaps and generates errors not previously observed during distributive synthesis, including clustered multiple substitutions and complex additions. Base substitution error rates are similar during filling of a <300- nucleotide gap and a 5-nucleotide gap, but are 2- to 10-fold lower for three mispairs during synthesis to fill a single- nucleotide gap. Single-base deletion error rates are similar during distributive and processive synthesis. These data suggest a model wherein the unique 8 kDa domain of pol beta influences fidelity during filling of short but not long gaps. Structure- function studies of this type probe several concepts for how DNA polymerases accurately copy DNA. It is our belief that they will improve our understanding of how the human genome is stably replicated and maintained, and how DNA adducts affect genome stability.